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Fgf and Esrrb integrate epigenetic and transcriptional networks that regulate self-renewal of trophoblast stem cells.

Latos PA, Goncalves A, Oxley D, Mohammed H, Turro E, Hemberger M - Nat Commun (2015)

Bottom Line: In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes.Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex.Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

View Article: PubMed Central - PubMed

Affiliation: 1] Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK [2] Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.

ABSTRACT
Esrrb (oestrogen-related receptor beta) is a transcription factor implicated in embryonic stem (ES) cell self-renewal, yet its knockout causes intrauterine lethality due to defects in trophoblast development. Here we show that in trophoblast stem (TS) cells, Esrrb is a downstream target of fibroblast growth factor (Fgf) signalling and is critical to drive TS cell self-renewal. In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes. To elucidate the mechanisms whereby Esrrb controls the expression of its targets, we characterized its TS cell-specific interactome using mass spectrometry. Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex. Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

No MeSH data available.


ChIP-seq analysis to identify Esrrb genome-wide occupancy in TS cells.(a) Anti-Esrrb ChIP followed by QPCR showing Esrrb binding to TS cell marker genes; the Ensa region serves as a negative control. Bars indicate average of three biological replicates±s.e.m. Statistical test: unpaired t-test with Welch's correction. (b) A Venn diagram showing the number of high-confidence Esrrb TS cell-specific peaks identified in five independent biological replicates of ChIP-seq experiments, ES cell-specific peaks29 and those overlapping in both stem lines. (c) Proportion of Esrrb ChIP-seq peaks overlapping genomic features in TS and ES cells. Peaks overlapping more than one type of genomic region were assigned to regions with the following priority: (1) promoters, (2) exons (3), introns, (4) downstream and (5) intergenic. (d) Twenty top terms of the GREAT ontology enrichments for TS cell-specific peaks of Esrrb. (e) Examples of Esrrb-binding profiles at the Eomes and Elf5 loci. (f) Luciferase reporter analysis of TS cells transiently transfected with putative Eomes (Eomes: pGL3-promoter-Eomes) or Elf5 (Elf5: pGL3-promoter-Elf5) enhancer constructs and controls (basic: pGL3-basic and promoter: pGL3-promoter). Bars show an average of four replicates±s.d., statistical test: unpaired t-test with Welch's correction. (g) Pie chart of genes deregulated on Esrrb knockdown (posterior probability >0.6) that are also bound by Esrrb. (h) Motifs found by MEME and/or DREME and SpaMO to be overrepresented in the Esrrb peaks. (i) CentriMO plot of the positional distribution of the best-matched motifs.
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f3: ChIP-seq analysis to identify Esrrb genome-wide occupancy in TS cells.(a) Anti-Esrrb ChIP followed by QPCR showing Esrrb binding to TS cell marker genes; the Ensa region serves as a negative control. Bars indicate average of three biological replicates±s.e.m. Statistical test: unpaired t-test with Welch's correction. (b) A Venn diagram showing the number of high-confidence Esrrb TS cell-specific peaks identified in five independent biological replicates of ChIP-seq experiments, ES cell-specific peaks29 and those overlapping in both stem lines. (c) Proportion of Esrrb ChIP-seq peaks overlapping genomic features in TS and ES cells. Peaks overlapping more than one type of genomic region were assigned to regions with the following priority: (1) promoters, (2) exons (3), introns, (4) downstream and (5) intergenic. (d) Twenty top terms of the GREAT ontology enrichments for TS cell-specific peaks of Esrrb. (e) Examples of Esrrb-binding profiles at the Eomes and Elf5 loci. (f) Luciferase reporter analysis of TS cells transiently transfected with putative Eomes (Eomes: pGL3-promoter-Eomes) or Elf5 (Elf5: pGL3-promoter-Elf5) enhancer constructs and controls (basic: pGL3-basic and promoter: pGL3-promoter). Bars show an average of four replicates±s.d., statistical test: unpaired t-test with Welch's correction. (g) Pie chart of genes deregulated on Esrrb knockdown (posterior probability >0.6) that are also bound by Esrrb. (h) Motifs found by MEME and/or DREME and SpaMO to be overrepresented in the Esrrb peaks. (i) CentriMO plot of the positional distribution of the best-matched motifs.

Mentions: To explore whether Esrrb directly regulates the key TS cell genes, we performed chromatin immunoprecipitation (ChIP) followed by QPCR and found extensive binding on putative transcriptional regulatory regions of Elf5, Eomes, Esrrb, Sox2, Bmp4, Cdx2 and Tfap2c (Fig. 3a). To obtain a comprehensive global overview of the binding sites of Esrrb in TS cells, we carried out ChIP followed by high-throughput sequencing (ChIP-seq) and compared these data to the binding profile of Esrrb in ES cells where it plays a well-appreciated role in maintaining pluripotency29. We identified 14507 Esrrb-binding sites in TS cells (Fig. 3b; Supplementary Data 4). Globally, these sites were predominantly found at intronic and intergenic regions (Fig. 3c), similar in feature distribution to that observed in ES cells. However, their precise location exhibited only a partial (3,027) overlap with those in ES cells (Fig. 3b; Supplementary Data 3). The markedly different Esrrb-binding profile between ES and TS cells was exemplified by a significant enrichment of genes involved in trophectodermal differentiation and placental development among the TS cell-specific peaks compared with the ES cell-specific peaks (Fig. 3d; Supplementary Fig. 3a). These results suggest that context-dependent binding of Esrrb is linked to specific developmental processes. Notably, we identified Esrrb binding at principally all known core TS cell genes, including itself, implying that Esrrb has a self-reinforcing function similar to that ascribed to many pluripotency genes in ES cells (Fig. 3e; Supplementary Fig. 3b).


Fgf and Esrrb integrate epigenetic and transcriptional networks that regulate self-renewal of trophoblast stem cells.

Latos PA, Goncalves A, Oxley D, Mohammed H, Turro E, Hemberger M - Nat Commun (2015)

ChIP-seq analysis to identify Esrrb genome-wide occupancy in TS cells.(a) Anti-Esrrb ChIP followed by QPCR showing Esrrb binding to TS cell marker genes; the Ensa region serves as a negative control. Bars indicate average of three biological replicates±s.e.m. Statistical test: unpaired t-test with Welch's correction. (b) A Venn diagram showing the number of high-confidence Esrrb TS cell-specific peaks identified in five independent biological replicates of ChIP-seq experiments, ES cell-specific peaks29 and those overlapping in both stem lines. (c) Proportion of Esrrb ChIP-seq peaks overlapping genomic features in TS and ES cells. Peaks overlapping more than one type of genomic region were assigned to regions with the following priority: (1) promoters, (2) exons (3), introns, (4) downstream and (5) intergenic. (d) Twenty top terms of the GREAT ontology enrichments for TS cell-specific peaks of Esrrb. (e) Examples of Esrrb-binding profiles at the Eomes and Elf5 loci. (f) Luciferase reporter analysis of TS cells transiently transfected with putative Eomes (Eomes: pGL3-promoter-Eomes) or Elf5 (Elf5: pGL3-promoter-Elf5) enhancer constructs and controls (basic: pGL3-basic and promoter: pGL3-promoter). Bars show an average of four replicates±s.d., statistical test: unpaired t-test with Welch's correction. (g) Pie chart of genes deregulated on Esrrb knockdown (posterior probability >0.6) that are also bound by Esrrb. (h) Motifs found by MEME and/or DREME and SpaMO to be overrepresented in the Esrrb peaks. (i) CentriMO plot of the positional distribution of the best-matched motifs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: ChIP-seq analysis to identify Esrrb genome-wide occupancy in TS cells.(a) Anti-Esrrb ChIP followed by QPCR showing Esrrb binding to TS cell marker genes; the Ensa region serves as a negative control. Bars indicate average of three biological replicates±s.e.m. Statistical test: unpaired t-test with Welch's correction. (b) A Venn diagram showing the number of high-confidence Esrrb TS cell-specific peaks identified in five independent biological replicates of ChIP-seq experiments, ES cell-specific peaks29 and those overlapping in both stem lines. (c) Proportion of Esrrb ChIP-seq peaks overlapping genomic features in TS and ES cells. Peaks overlapping more than one type of genomic region were assigned to regions with the following priority: (1) promoters, (2) exons (3), introns, (4) downstream and (5) intergenic. (d) Twenty top terms of the GREAT ontology enrichments for TS cell-specific peaks of Esrrb. (e) Examples of Esrrb-binding profiles at the Eomes and Elf5 loci. (f) Luciferase reporter analysis of TS cells transiently transfected with putative Eomes (Eomes: pGL3-promoter-Eomes) or Elf5 (Elf5: pGL3-promoter-Elf5) enhancer constructs and controls (basic: pGL3-basic and promoter: pGL3-promoter). Bars show an average of four replicates±s.d., statistical test: unpaired t-test with Welch's correction. (g) Pie chart of genes deregulated on Esrrb knockdown (posterior probability >0.6) that are also bound by Esrrb. (h) Motifs found by MEME and/or DREME and SpaMO to be overrepresented in the Esrrb peaks. (i) CentriMO plot of the positional distribution of the best-matched motifs.
Mentions: To explore whether Esrrb directly regulates the key TS cell genes, we performed chromatin immunoprecipitation (ChIP) followed by QPCR and found extensive binding on putative transcriptional regulatory regions of Elf5, Eomes, Esrrb, Sox2, Bmp4, Cdx2 and Tfap2c (Fig. 3a). To obtain a comprehensive global overview of the binding sites of Esrrb in TS cells, we carried out ChIP followed by high-throughput sequencing (ChIP-seq) and compared these data to the binding profile of Esrrb in ES cells where it plays a well-appreciated role in maintaining pluripotency29. We identified 14507 Esrrb-binding sites in TS cells (Fig. 3b; Supplementary Data 4). Globally, these sites were predominantly found at intronic and intergenic regions (Fig. 3c), similar in feature distribution to that observed in ES cells. However, their precise location exhibited only a partial (3,027) overlap with those in ES cells (Fig. 3b; Supplementary Data 3). The markedly different Esrrb-binding profile between ES and TS cells was exemplified by a significant enrichment of genes involved in trophectodermal differentiation and placental development among the TS cell-specific peaks compared with the ES cell-specific peaks (Fig. 3d; Supplementary Fig. 3a). These results suggest that context-dependent binding of Esrrb is linked to specific developmental processes. Notably, we identified Esrrb binding at principally all known core TS cell genes, including itself, implying that Esrrb has a self-reinforcing function similar to that ascribed to many pluripotency genes in ES cells (Fig. 3e; Supplementary Fig. 3b).

Bottom Line: In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes.Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex.Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

View Article: PubMed Central - PubMed

Affiliation: 1] Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK [2] Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.

ABSTRACT
Esrrb (oestrogen-related receptor beta) is a transcription factor implicated in embryonic stem (ES) cell self-renewal, yet its knockout causes intrauterine lethality due to defects in trophoblast development. Here we show that in trophoblast stem (TS) cells, Esrrb is a downstream target of fibroblast growth factor (Fgf) signalling and is critical to drive TS cell self-renewal. In contrast to its occupancy of pluripotency-associated loci in ES cells, Esrrb sustains the stemness of TS cells by direct binding and regulation of TS cell-specific transcription factors including Elf5 and Eomes. To elucidate the mechanisms whereby Esrrb controls the expression of its targets, we characterized its TS cell-specific interactome using mass spectrometry. Unlike in ES cells, Esrrb interacts in TS cells with the histone demethylase Lsd1 and with the RNA Polymerase II-associated Integrator complex. Our findings provide new insights into both the general and context-dependent wiring of transcription factor networks in stem cells by master transcription factors.

No MeSH data available.